CCR2+ Inflammatory Dendritic Cells and Translocation of Antigen by Type III Secretion Are Required for the Exceptionally Large CD8+ T Cell Response to the Protective YopE69-77 Epitope during Yersinia Infection

During Yersinia pseudotuberculosis infection of C57BL/6 mice, an exceptionally large CD8⁺ T cell response to a protective epitope in the type III secretion system effector YopE is produced. At the peak of the response, up to 50% of splenic CD8⁺ T cells recognize the epitope YopE_69-77. The features of the interaction between pathogen and host that result in this large CD8⁺ T cell response are unknown. Here, we used Y. pseudotuberculosis strains defective for production, secretion and/or translocation of YopE to infect wild-type or mutant mice deficient in specific dendritic cells (DCs). Bacterial colonization of organs and translocation of YopE into spleen cells was measured, and flow cytometry and tetramer staining were used to characterize the cellular immune response. We show that the splenic YopE_69-77-specific CD8⁺ T cells generated during the large response are polyclonal and are produced by a “translocation-dependent” pathway that requires injection of YopE into host cell cytosol. Additionally, a smaller YopE_69-77-specific CD8⁺ T cell response (~10% of the large expansion) can be generated in a “translocation-independent” pathway in which CD8α⁺ DCs cross present secreted YopE. CCR2-expressing inflammatory DCs were required for the large YopE_69-77-specific CD8⁺ T cell expansion because this response was significantly reduced in Ccr2^-/- mice, YopE was translocated into inflammatory DCs in vivo, inflammatory DCs purified from infected spleens activated YopE_69-77-specific CD8⁺ T cells ex vivo and promoted the expansion of YopE_69-77-specific CD8⁺ T cells in infected Ccr2^-/- mice after adoptive transfer. A requirement for inflammatory DCs in producing a protective CD8⁺ T cell response to a bacterial antigen has not previously been demonstrated. Therefore, the production of YopE_69-77-specific CD8⁺ T cells by inflammatory DCs that are injected with YopE during Y. pseudotuberculosis infection represents a novel mechanism for generating a massive and protective adaptive immune response.     

Construction of High Density Sweet Cherry (Prunus avium L.) Linkage Maps Using Microsatellite Markers and SNPs Detected by Genotyping-by-Sequencing (GBS)

Linkage maps are valuable tools in genetic and genomic studies. For sweet cherry, linkage maps have been constructed using mainly microsatellite markers (SSRs) and, recently, using single nucleotide polymorphism markers (SNPs) from a cherry 6K SNP array. Genotyping-by-sequencing (GBS), a new methodology based on high-throughput sequencing, holds great promise for identification of high number of SNPs and construction of high density linkage maps. In this study, GBS was used to identify SNPs from an intra-specific sweet cherry cross. A total of 8,476 high quality SNPs were selected for mapping. The physical position for each SNP was determined using the peach genome, Peach v1.0, as reference, and a homogeneous distribution of markers along the eight peach scaffolds was obtained. On average, 65.6% of the SNPs were present in genic regions and 49.8% were located in exonic regions. In addition to the SNPs, a group of SSRs was also used for construction of linkage maps. Parental and consensus high density maps were constructed by genotyping 166 siblings from a ‘Rainier’ x ‘Rivedel’ (Ra x Ri) cross. Using Ra x Ri population, 462, 489 and 985 markers were mapped into eight linkage groups in ‘Rainier’, ‘Rivedel’ and the Ra x Ri map, respectively, with 80% of mapped SNPs located in genic regions. Obtained maps spanned 549.5, 582.6 and 731.3 cM for ‘Rainier’, ‘Rivedel’ and consensus maps, respectively, with an average distance of 1.2 cM between adjacent markers for both ‘Rainier’ and ‘Rivedel’ maps and of 0.7 cM for Ra x Ri map. High synteny and co-linearity was observed between obtained maps and with Peach v1.0. These new high density linkage maps provide valuable information on the sweet cherry genome, and serve as the basis for identification of QTLs and genes relevant for the breeding of the species.     

Invertebrate Metacommunity Structure and Dynamics in an Andean Glacial Stream Network Facing Climate Change

Under the ongoing climate change, understanding the mechanisms structuring the spatial distribution of aquatic species in glacial stream networks is of critical importance to predict the response of aquatic biodiversity in the face of glacier melting. In this study, we propose to use metacommunity theory as a conceptual framework to better understand how river network structure influences the spatial organization of aquatic communities in glacierized catchments. At 51 stream sites in an Andean glacierized catchment (Ecuador), we sampled benthic macroinvertebrates, measured physico-chemical and food resource conditions, and calculated geographical, altitudinal and glaciality distances among all sites. Using partial redundancy analysis, we partitioned community variation to evaluate the relative strength of environmental conditions (e.g., glaciality, food resource) vs. spatial processes (e.g., overland, watercourse, and downstream directional dispersal) in organizing the aquatic metacommunity. Results revealed that both environmental and spatial variables significantly explained community variation among sites. Among all environmental variables, the glacial influence component best explained community variation. Overland spatial variables based on geographical and altitudinal distances significantly affected community variation. Watercourse spatial variables based on glaciality distances had a unique significant effect on community variation. Within alpine catchment, glacial meltwater affects macroinvertebrate metacommunity structure in many ways. Indeed, the harsh environmental conditions characterizing glacial influence not only constitute the primary environmental filter but also, limit water-borne macroinvertebrate dispersal. Therefore, glacier runoff acts as an aquatic dispersal barrier, isolating species in headwater streams, and preventing non-adapted species to colonize throughout the entire stream network. Under a scenario of glacier runoff decrease, we expect a reduction in both environmental filtering and dispersal limitation, inducing a taxonomic homogenization of the aquatic fauna in glacierized catchments as well as the extinction of specialized species in headwater groundwater and glacier-fed streams, and consequently an irreversible reduction in regional diversity.     

Phenotype and growth behavior of residual β-catenin-positive hepatocytes in livers of β-catenin-deficient mice

Signaling through the Wnt/β-catenin pathway is a crucial determinant of hepatic zonal gene expression, liver development, regeneration, and tumorigenesis. Transgenic mice with hepatocyte-specific knockout of Ctnnb1 (encoding β-catenin) have proven their usefulness in elucidating these processes. We now found that a small number of hepatocytes escape the Cre-mediated gene knockout in that mouse model. The remaining β-catenin-positive hepatocytes showed approximately 25% higher cell volumes compared to the β-catenin-negative cells and exhibited a marker protein expression profile similar to that of normal perivenous hepatocytes or hepatoma cells with mutationally activated β-catenin. Surprisingly, the expression pattern was observed independent of the cell's position within the liver lobule, suggesting a malfunction of physiological periportal repression of perivenously expressed genes in β-catenin-deficient liver. Clusters of β-catenin-expressing hepatocytes lacked expression of the gap junction proteins Connexin 26 and 32. Nonetheless, β-catenin-positive hepatocytes had no striking proliferative advantage, but started to grow out on treatment with phenobarbital, a tumor-promoting agent known to facilitate the formation of mouse liver adenoma with activating mutations of Ctnnb1. Progressive re-population of Ctnnb1 knockout livers with wild-type hepatocytes was seen in aged mice with a pre-cirrhotic phenotype. In these large clusters of β-catenin-expressing hepatocytes, perivenous-specific gene expression was re-established. In summary, our data demonstrate that the zone-specificity of a hepatocyte's gene expression profile is dependent on the presence of β-catenin, and that β-catenin provides a proliferative advantage to hepatocytes when promoted with phenobarbital, or in a pre-cirrhotic environment.     

A BOD monitoring disposable reactor with alginate-entrapped bacteria

Biochemical oxygen demand (BOD) is a measure of the amount of dissolved oxygen that is required for the biochemical oxidation of the organic compounds in 5 days. New biosensor-based methods have been conducted for a faster determination of BOD. In this study, a mathematical model to evaluate the feasibility of using a BOD sensor, based on disposable alginate-entrapped bacteria, for monitoring BOD in situ was applied. The model considers the influences of alginate bead size and bacterial concentration. The disposable biosensor can be adapted according to specific requirements depending on the organic load contained in the wastewater. Using Klein and Washausen parameter in a Lineweaver–Burk plot, the glucose diffusivity was calculated in 6.4 × 10⁻¹⁰ (m²/s) for beads of 1 mm in diameter and slight diffusion restrictions were observed (n = 0.85). Experimental results showed a correlation (p < 0.05) between the respirometric peak and the standard BOD test. The biosensor response was representative of BOD.     

Patterns of genetic diversity in colonizing plant species: Nassauvia lagascae var. lanata (Asteraceae: Mutisieae) on Volcan Lonquimay, Chile

The effect of colonization on the distribution of genetic diversity within and among populations in relation to species characteristics remains an open empirical question. The objective of this study was to contrast genetic diversity within and among established and colonizing populations of Nassauvia lagascae var. lanata on Volcán Lonquimay (Araucanía Region, Chile), which erupted on 25 December 1988, and relate genetic diversity to biological characteristics of the populations. We analyzed a total of 240 individuals from 15 populations distributed along the Andes Cordillera using AFLP and obtained a total of 307 AFLP bands, of which 97.7% are polymorphic. Values of population differentiation (F(ST)) did not differ significantly among established and colonizing populations, but colonizing populations did have reduced levels of genetic divergence (as indicated by private and rare bands) and genetic variation (e.g., Shannon index). We conclude that a founder effect through limited numbers of founding propagules derived from nearby source populations has not yet been compensated for by subsequent population growth and migration. Low rates of secondary dispersal via running water, kin-structure within populations, and slow population growth seem to contribute to the slow recovery of genetic diversity.     

Trans platinum complexes design: one novel water soluble oxime derivative that contains aliphatic amines in trans configuration

In an attempt to design new antitumoral drugs based on transplatin complexes, we determined the experimental conditions for the preparation of trans-[Pt((CH(3))(2)CNOH)((CH(3))(2)CHNH(2))Cl(2)], and solved the crystal structure. The cytotoxicity of the novel complex, the cis counterpart, cisplatin, and a trans complex with aliphatic amines, as well as the capacity of some of these complexes to cause either apoptotic or necrotic cell death, was comparatively examined in NRK-52E rat renal tubular cells and HepG2 human hepatoma cells. The results indicate that the oxime complex with trans geometry, but not the one with cis geometry, causes death by apoptosis, making the complex potentially suitable for therapeutic purposes. However cytotoxicity values are higher in the case of cis geometry than in trans geometry in both tumoral and non-tumoral cell lines.